Current methods for determining fecal contamination of recreational waters rely on the culture of bacterial indicators and require at least 24 hours to determine whether the water is unsafe for use. By the time monitoring results are available, exposures have already occurred. New methods are needed that will allow near real-time determination of water quality, such that public notifications can be made and hazardous exposures avoided. With assistance from the U.S. Geological Survey Laboratory in Porter IN, the U.S. EPA, National Exposure Research Laboratory has conducted a two year pilot study to evaluate the use of quantitative PCR (QPCR) analysis for measuring water-borne fecal indicator microorganisms at two recreational beaches on Lake Michigan.

A rapid, simple and generally applicable method for the recovery of total DNA from various microorganisms in water samples has been developed. The method involves filtration of water samples on polycarbonate membrane filters and disruption of the collected cells directly on the membranes by glass bead milling. Recovered DNAs are subjected to QPCR analysis using the TaqManTM PCR product detection system in a real time PCR product detection instrument. Procedures have also been developed for the use of cycle threshold (CT) values generated by the instrument to enumerate cells in the water samples. The approach is based on the comparative cycle threshold (CT) method, which employs an arithmetic formula to determine target sequence quantities in DNA extracts from test samples relative to those in similarly-prepared DNA extracts from calibrator samples containing a known quantity of target organism cells. Assay CT values for a DNA sequence from an exogenous reference organism, added in equal quantities to both the test and calibrator samples before extraction, are used to normalize results for differences in the amount of total DNA added to each reaction (e.g., caused by differences in DNA extraction efficiency between samples) or to signal potential PCR inhibition in test samples. The entire analysis process can be performed in approximately two to three hours.

Target DNA sequences for QPCR detection in the pilot study included the large subunit ribosomal RNA gene of Enterococcus spp. and the small subunit ribosomal RNA gene of Bacteroides spp. Tests with pure cultures of representative species within these two genera gave extrapolated detection limits of approximately two cells per sample for the Enterococcus assay and 25 cells per sample for the Bacteroides assay. Tests on a subset of the Lake Michigan water samples spiked with ~1000 cells of Enterococcus cells gave an overall mean value of 0.96 for the ratio of measured to added cells and a 95% occurrence range for individual sample ratios of ~0.3 to 3, based on analyses of three replicates of each sample. Mean QPCR-measured quantities of native enterococci in the 100 ml water samples ranged from less than 10 to ~1000 cells whereas mean quantities of Bacteroides ranged from less than 100 to ~100,000 cells. DNA extracts of the water samples were routinely diluted 10-fold prior to analysis to eliminate the effects of PCR inhibitors. The distributions of QPCR-measured cell quantities of both Enterococcus and Bacteroides in the water samples paralleled those of culturable enterococci measured in corresponding water samples by the currently accepted mEI filter plating method.

These results indicate that the QPCR method has the potential to detect a broad range of fecal indicator densities in recreational water samples. Past findings of a correlation between the quantities of culturable enterococci in water samples and illness rates among bathers have provided the basis for establishing recreational water quality guidelines. New studies will be initiated this summer to establish whether similar correlations exist between fecal indicator measurements by QPCR and other rapid methods and rates of illness among bathers.